The walls of plant cells are formed of cellulose, hemicellulose, and lignin polymers, which contribute to cell wall structure and rigidity. These complex carbohydrates act as a physical barrier to infection by plant pathogens, and are necessary to prevent dehydration and maintain physical integrity. Xylan, a polymeric chain of xylose carbohydrate monomers linked by successive .beta.-1,4 xylose linkages, constitutes a significant portion of plant hemicellulose. Xylan contributes to the tensile strength of plant secondary walls, and functions to limit the accessibility of cellulases to the cell wall, thereby preventing damage to the plant surface by challenge of plant pathogens.
The degradation of plant tissues involves, in part, the hydrolysis of xylan by enzymes produced by fibrolytic bacteria and fungi. The physical structure of xylan is complex, and its degradation is accomplished by a consortium of hydrolytic enzymes. Substituent-free .beta.-1,4 xylan polymers are digested into shorter oligoxylan fragments by endoxylanlolytic enzymes (EC 3.2.1.8), and the resulting oligoxylan fragments are subsequently degraded into xylose monomers by .beta.-xylosidases (EC 3.2.1.37) (Bieley, 1985). However, the hydrolysis of xylan is complicated by the presence of various substituents (such as acetyl, arabinosyl, and glucuranosyl) along the elongated .beta.-1,4 xylose chains. The prevalence of various substituent groups on xylan polymers varies amongst differing plant types. Xylan derived from grasses appear to predominantly contain arabinose constituents, whereas xylan polymers derived from hardwoods contain acetyl and arabinose substituents. These substituents attached to the .beta.-1,4 xylan backbone obstruct endoxylanolytic hydrolysis by steric impediment of the xylanase enzymes. Xylanolytic degradation is therefore effected by xylanases in concert with enzymes such as .alpha.-L-arabinofuranosidases (EC 3.2.1.55) and acetylesterases (EC 3.1.1.6), which cleave substituents from the xylan polymer.
Xylanolytic enzymes have numerous applications in industry and biotechnology. Such applications include the supplementation of xylanases in biopulping processes. For example, pulp quality may be improved by the enzymatic removal of xylan from woody plant tissue. Predigestion of forage crops with cellulases and xylanases can improve nutritional availability of feed carbohydrates and enhance fermentative composting. Furthermore, xylanases may be employed to degrade agricultural wastes, thereby reducing organic waste disposal in landfill sites. Xylanases have also been used to improve the quality of flour in baked goods. Because xylan represents a considerable reserve of nutritive sugar sequestered in plant biomass, xylanases are important in ruminal digestion of forage fiber, where the release of xylose monomers may provide a nutritive source for non-xylanolytic ruminal microflora.
Given the industrial importance of xylanases, there is great interest in isolating new, highly active xylanases that can be produced inexpensively. Ideally, such enzymes should be active over broad temperature and pH ranges, and are preferably functional in both prokaryotic and eukaryotic expression systems.
A range of microorganisms produce xylanases, and from these a number of xylanase genes have been isolated. Xylanase genes can be categorized into two main families, 10 (clan GH-A, previously F family of glycosyl hydrolases) and 11 (clan GH-C, previously G family of glycosyl hydrolases) (Henrissat et al., 1989; Henrissat and Bairoch, 1993). Many microbial xylanases have been identified in the literature. Fungal xylanases originating from Aspergillus species (Arai et al. 1998, Ito et al. 1992), Emericella nidulans (MacCabe et al. 1996, previously identified as Aspergillus nidulans), and Penicillium chrysogenum (Haas et al. 1993) contain a catalytic domain which appears to be conserved among these species and encode polypeptides containing between 327 and 347 amino acids. Xylanases originating from Fursarium oxysporum (Ruiz-Roldan et al., 1998; Sheppard et al., 1994) contain a conserved catalytic domain and, additionally, a cellulose binding domain and a linker or hinge domain attaching the cellulose binding domain to the catalytic domain. With the additional domains, the polypeptides contain 384 amino acids. U.S. Pat. No. 5,763,254 to Woldike et al. discloses the DNA sequences and derived amino acid sequences of F. oxysporum C-family cellobiohydrolase, F. oxysporum F-family cellulase, F. oxysporum C-family endoglucanase, and Humicola insolens endoglucanase 1, each of which possess a carbohydrate binding domain, a linker region, and a catalytic domain.
The fungus Coniothyrium minitans Campbell is a mycoparasite capable of attacking the fungal plant pathogen Sclerotinia sclerotiorum (Lib) de Bary (Huang and Hoes, 1976), resulting in the degradation of sclerotia (Huang and Kokko, 1987) and hyphae (Huang and Kokko, 1988) of the pathogen. Jones et al. (1974) reported endo- and exo-1,3-.beta.-glucanase activity in C. minitans. International application No. PCT/CA98/00668 discloses strains of C. minitans having high levels of .beta.-glucanase activity when grown by liquid or solid state fermentation. However, the prior art has not looked to C. minitans as a source for the isolation of xylanase genes.